PROJECT SUMMARY The placenta has a unique, critical, functional role during development in regulating the intrauterine environment and fetal development. This project is specifically focused on better characterizing the epigenome of the placenta, as these functionally relevant molecular features may elucidate mechanisms by which environmental exposures influence offspring development and may also reveal biomarkers of perinatal exposures and future health risk or protection. The presence of 5-methylcytosine (5mC) within a promoter region is conventionally associated with a reduction in gene expression, but our understanding of gene expression control has been broadening, in particular with the discovery of 5-hydroxymethylcytosine (5hmC), an oxidative product of the process of DNA demethylation. Data from our own small pilot study in placenta and a growing body of literature in other tissues is suggesting a potential for 5hmC to function not only as a cellular intermediate, but also as a stand-alone stable epigenetic modification which is overrepresented enhancer regions and is associated with increased gene expression. Traditional bisulfite-based techniques used to study 5mC, though, cannot distinguish between 5mC and 5hmC, and as these modifications may have distinct functional implications, understanding more precisely how these marks are distributed and related to functional effects can help to clarify the mechanistic way in which these modifications operate and link to environmental exposures and outcomes. We hypothesize that 5hmC in the placenta marks poised enhancer regions and can serve as a marker of actively transcribed regions of the placental genome. In addition, we hypothesize that the inter-individual differences in 5hmC extent are associated with environmental factors and infant outcomes. We will examine these hypotheses using employ state-of-the- art experimental and analytical approaches in a highly characterized subset of placenta samples (n=200) from the Rhode Island Child Health Study (RICHS) cohort, which has detailed epidemiologic, clinical, and demographic data on the mothers and infants involved, as well as biomarker-based assessments of trace metals exposures, and whole transcriptome expression data through RNAseq. These results will provide the most comprehensive, to date, delineation of the DNA cytosine modification landscape of the human placenta and its link to gene expression control, support the conceptual development of a larger project focused on the functional characterization of the placental epigenome and its role in the developmental origins of children's health, and inform novel hypotheses regarding both environmental drivers of variability in cytosine modification as well as its association with critical newborn health outcomes.